All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Plant sterols are naturally occurring substances present in the diet as minor components of vegetable oils. Plant sterols have a role in plants similar to that of cholesterol in mammals, e.g. forming cell membrane structures. In human nutrition, both plant sterols and plant stanols are effective in lowering total plasma cholesterol and LDL-cholesterol levels. The term “phytosterols” covers plant sterols and plant stanols.
Phytosterols are components of a normal diet, mainly coming from plant sources, e.g. vegetable oils, seeds, nuts and grain-based products. The typical consumption of plant sterols in Western diets ranges between 200 and 400 mg/d, whereas the intake of phytostanols, the saturated form of plant sterols, is negligible.
The consumption of higher amounts of plant sterols and plant stanols lowers blood cholesterol levels by inhibiting the absorption of dietary and endogenously-produced cholesterol from the small intestine. This inhibition is related to the similarity in physico-chemical properties of plant sterols and stanols to cholesterol.
The plant sterols/stanols are very poorly absorbable compounds. Studies on the absorption, distribution, metabolism and excretion have shown that plant sterols are poorly absorbed from the intestine (1-10%).
The hypocholesterolemic action of phytosterols has been recognized since the early 1950's [Jones et al. (1997) Can. J. Physiol. Pharmacol. 75, 217-227]. The blood cholesterol-lowering effect of plant sterols has been investigated in a large number of clinical trials involving over 2,400 subjects, using doses as high as 25 grams per day for durations as long as three years. No significant adverse effects have been observed throughout the decades of medically supervised clinical efficacy testing or the general clinical use of plant sterols. Furthermore, the drug Cytellin (primarily β-sitosterol) was prescribed for more than 20 years and had an excellent safety record. In addition, both plant sterols and plant stanols have been subjected to rigorous toxicological evaluation.
Phytosterols or phytostanols, as well as their fatty acid esters and other derivatives, are common products provided as dietary supplements or as functional ingredients in a variety of health promoting foods and beverages, as well as dietary supplements, all in the aim of reducing blood levels of total cholesterol, and especially of LDL (low density lipoprotein) cholesterol.
Hypercholesterolemia (high blood cholesterol), which can be treated or controlled by phytosterols, is a major risk factor of cardiovascular disorders (CVD). CVD is one of the leading causes of mortality worldwide. Hypercholesterolemia also causes the formation and accumulation of plaque deposits in the arteries, which results in atherosclerosis, the pathological process underlying coronary heart disease (CHD), which is one kind of cardiovascular disorders.
Type 2 diabetes and also the associated conditions known as diabesity and metabolic syndrome, are related to CVD, whose incidence has also dramatically increased over the last century. In most cases, cardiovascular disorders are enhanced and even triggered by such metabolic disorders or their onset and development is accelerated by said syndromes. These are also closely related to risk factors of abnormal blood lipid profile, especially high levels of total cholesterol and non-HDL cholesterol species, mainly LDL cholesterol.
People at risk to develop a metabolic syndrome, diabetes, or obesity, as well as CVD or CHD, or that are already classified as patients of said conditions are cautioned to monitor and control their blood lipids profile, especially high levels of blood cholesterol. One of the most efficient methods to address this risk factor common to so many health conditions and risks is the ingestion of plant sterols or stanols or their derivatives.
A potent method of treatment that has been used to address such risk factor was the supplementation of both statins and phytosterols. Both active ingredients address high levels of blood cholesterol, each using a different mechanism of action. The pharmaceutical ingredients based on statins inhibit the de novo synthesis of cholesterol, while phytosterols inhibit the absorption of dietary and endogenously-produced cholesterol. It has been demonstrated that consumption of plant stanol esters by patients taking statin therapy but that still have elevated LDL-cholesterol levels, was able to further reduce the total and LDL cholesterol levels by up to 12% and 17%, respectively [Blair et al. (2000) Am. J. Cardiol. 86, 46-52]. In addition, treating subjects with primary hypercholesterolemia with statins and phytosterol-ester containing foods produced a purely additive effect on LDL cholesterol, reduction [Simons et al. (2002) Am. J. Cardiol. 90, 737-740]. In fact, Simons et al. have concluded that the addition of phytosterol-esters to statins therapy offers an LDL cholesterol reduction equivalent to doubling the dose of statins. Similar results were exhibited by others [Vuorio et al. (2000) Arterioscler. Thromb. Vasc. Biol. 500-506].
Post-menopausal females demonstrate several typical symptoms, like hot flashes, night sweats, sleeplessness, vaginal dryness, and disorders such as osteoporosis, CVD and central nervous system related alterations (anxiety and mood swings, depression and cognitive decline). With regards to the CVD complications in this population, it is well established that the rate of myocardial infarction increases by 3 fold and that cerebrovascular disease rises rapidly. It is believed that estrogen plays role in protecting endothelial cell wall and possibly protecting against thrombosis, hence the aforementioned symptoms following its deficiency. Hormone replacement therapy (HRT) has a clear role in the treatment of menopausal symptoms and osteoporosis. However, recent findings from Women Health Initiative (WHI) showed that taking HRT increases a woman's risk for heart disease, stroke, and pulmonary embolism. Recently, postmenopausal women (aged 50-55) consuming margarine-containing plant stanols for 6 weeks demonstrated a marked reduction of LDL cholesterol versus baseline, while HDL-cholesterol levels were increased, so the LDL/HDL cholesterol ratio was reduced [Gylling and Miettinen (1999) Metabolism 48:575-580]. However, following the different stanol interventions there was a notable decrease in □- and □-carotene concentrations while retinal, vitamin D and lipid standardized □-tocopherol levels were comparable to baseline values.
Phytosterols have also been connected and shown to have anti-cancer effects. Epidemiologic and experimental studies suggest that dietary phytosterols may offer protection from the most common cancers in Western societies, such as colon, breast and prostate cancer. Populations at low breast cancer risk consume more dietary phytosterols than those at high risk. Several in vitro studies have suggested that phytosterols are toxic to human breast cancer and prostate cancer cells. In vivo animal experiments have demonstrated the inhibitory effect of phytosterols on the incidence, growth and metastasis of colon tumors, breast, and prostate cancer [Awad, A. B. and Fink C. S. (2000) J. Nutr. 130:2127-2130; Moreaua, R. A. et al. (2002) Progress in Lipid Research 41:457-500]. Although there is evidence that phytosterols have anti-carcinogenic properties, the mechanism by which they inhibit tumor growth is not yet known. However, possible mechanisms by which phytosterols offer this protection include direct effect on membrane structure and function of tumor and host tissue, signal transduction pathways that regulate tumor growth and apoptosis [Awad and Fink (2000) id ibid].
One of the major risk factors to CVD and CHD, as well as of metabolic disorders such as diabetes type 2, is oxidative stress, a relative increase in free radicals. These are responsible, for example, for the increased formation of oxidized-LDL, which when found at relatively high levels, is responsible to the formation of foam cells, the hallmark of early atherosclerosis. Free radicals may be found at high levels in the blood and other tissues due to a variety of causes, some environmental, such as increased pollution, or related to poor nutrition, due to ingestion of pro-oxidative nutrients, or to insufficient ingestion of different anti-oxidative nutrients. In addition, elevated free radicals levels are common in metabolic conditions such as type 2 diabetes, diabesity and metabolic syndrome, accompanied with sustained blood hyperglycemic state. Oxidative stress can be treated or may be even prevented by a variety of active ingredients possessing anti-oxidative activity, basically neutralizing free radicals found in the body.
Phytosterols could have a possible role in prevention of inflammation-related conditions or disorders, including lymphocyte proliferative responses, pulmonary tuberculosis, human immunodeficiency virus (HIV), stress-induced immune suppression, rheumatoid arthritis, and allergic rhinitis/sinusitis [Bouic (2001) Opin Clin Nutr. Metab. Care 4:471-475]. The mechanisms by which plant sterols display their anti-inflammatory activity are thought to include inhibition of secretion of inflammatory mediators such as interleukin-6, and tumor necrosis factor-□ by monocytes; however most of the work has been conducted using animals models and therefore the specific mechanism remain to be elucidated.
Phytosterols also present anti-ulcer activity, protecting against Helicobacter pylori infection. In a recent study, phytosterol esters, but not sterols, in horse gram (an herb in the genus Dolichos cultivated in India for food and fodder) were protective in a pyloric ligation model of ulcer [Jayaraj et al. (2003) Phytother Res 17:391-398]. Together with sterols and phospholipids, these food lipids in diets may account for the low prevalence of duodenal ulcer in certain geographical areas, despite a uniformly high prevalence of Helicobacter pylori infection.
Any treatment addressing said disorders and/or conditions needs to be accompanied by a healthy and balanced nutrition. This nutrition should of course provide the patient with all the essential nutrients, among these vitamins. Many of the vitamins are lipophilic, and among those important to such patients are beta-carotene, vitamin A, vitamin D and vitamin E. These vitamins also possess anti-oxidative activity which is important to protect the body from oxidative stress and perhaps prevent the damages of free radical activities. Beta carotene is one of the most important and abundant of the carotenes, a portion of which the liver converts to vitamin A. Beta carotene is a powerful antioxidant with properties that can contribute to reducing cancer and heart disease. It's found in carrots and many colorful vegetables. Vitamin A, also known as retinol, is a carotenoid vitamin (see Scheme 1). Vitamin A is produced in the liver from different provitamin carotenoids, especially from beta-carotene, the most important provitamin A, and an anti-oxidant in itself. Vitamin D is acquired from the diet or produced in the skin and is biologically inactive. It must be metabolized by the liver to produce the 25-hydroxyvitamin D (25-OH-D). However, this compound is also biologically inactive under physiological conditions and must be activated by the kidney to produce the final vitamin D hormone, 1,25-dihydroxyvitamin D3. This hormonal form of vitamin D is important in calcium metabolism to form strong bones and teeth and prevent rickets, osteomalacia and osteoporosis. Vitamin E, also known as alpha-tocopherol, is mainly found in plant sources, and is also a potent anti-oxidant. This lipophilic vitamin is usually accompanied by other derivatives of the tocopherols skeleton, such as beta and gamma tocopherols.
While phytosterols and phytostanols, and their fatty acid esters, are one of the most efficient methods of treatment of the hypercholesterolemia risk factor, either alone or in combination with statins or other ingredients, recent studies have shown that the use of these phyto-ingredients may cause adverse effects. The primary concern regarding phytosterol supplementation is the effect it may have on the absorption and circulating levels of lipid soluble vitamins and carotenoids. Dietary phytosterols inhibit the absorption of dietary and biliary cholesterol, which in turn decreases the absorption of carotenoids and lipid soluble vitamins [Noakes et al. (2002) Am. J. Clin. Nutr. 75(1):79-86]. This presents a concern because, as mentioned above, there are many health benefits associated with dietary consumption of carotenoids and vitamins [Hendriks et al. (1999) Eur. J. Clin. Nutr. 53(4): 319-327].
Moreover, several clinical trials were identified in which the consumption of phytosterols was reported to induce a significant decrease in plasma carotenoid levels [Katan et al. (2003) Mayo Clin. Proc. 78(8): 965-978]. These decreases followed the consumption of spread or oils containing between 0.83 and 3.6 g phytosterols/day for periods of 3 to 52 weeks. Recently, the bioavailability of beta-carotene and alpha-tocopherol was reported to decrease in mildly overweight male volunteers following the consumption of 2.2 g phytosterols/day for a year [Richelle et al. (2004) Am J. Clin. Nutr. 80(1):171-177]. However, in this trial, plant sterol esters reduced the bioavailability of beta-carotene and alpha-tocopherol more than free plant sterols did. The reduction in beta-carotene levels was as high as 50%. Although conventional phytosterol esters (esterified with n-6 polyunsaturated fatty acids, such as soybean oil and sunflower oil fatty acids) are inferior to free phytosterols in their effect on vitamin absorption, they have the advantage of increased solubility, which is important for the process of phytosterol incorporation into micelles and inhibition of cholesterol absorption [Ostlund R E, Jr. (2004) Phytosterols and cholesterol metabolism. Curr Opin Lipidol 15, 37-41].
In addition, while the beneficial effect of phytosterol and statins combination therapy was demonstrated [Vuorio (2000) id ibid.], monitoring the blood levels of vitamin A, vitamin E, as well as of alpha- and beta-carotene, revealed that this treatment has resulted in reduction of serum levels of all vitamins and pro-vitamins, ranging from 10 to 50%. These effects were especially pronounced in children undergoing treatment due to a genetic condition [see review in Berger et al. (2004) Lipids Health Dis. 3:5-24].
The European Union Scientific Committee on Food (SCF) has concluded that marketing of foods containing phytosterols should be accompanied by an investigation of possible adverse health effects, including among others the effects on plasma beta-carotene levels. The UK Food Advisory Committee (FAC) has recommended that consumers should be informed that phytosterol ester-containing products are not nutritionally appropriate for young children and breast feeding women as they do not need to reduce their blood cholesterol levels and there is a possibility of affecting vitamin A levels. The Scientific Panel on Dietetic Products, Nutrition and Allergies of the European Food Safety Authority (EFSA) has emphasized the need for risk management measures in order to minimize the likelihood of a daily intake exceeding 3 g phytosterols/phytostanols, the provision of appropriate information to consumers regarding the need for regular consumption of fruits and vegetables to address the potential beta-carotene lowering effect of the product.
Lichtenstein and colleagues [Lichtenstein et al. (2001) Circulation. 103: 1177-1179] have pointed potential risks associated with the use of plant stanol/sterol ester-containing fats, among them observations of decreased levels of alpha and beta carotene, alpha-tocopherol, and/or lycopene in the plasma, as a result of the consumption of foods containing both stanol esters and sterol esters.
Since food products containing plant sterols are likely to be shared during meals by all family members, the potential for intake by non-hypercholesterolemic individuals is significant. Thus, the American Heart Association recommended that further studies and large-scale monitoring be undertaken to determine the long-term safety of plant sterol/stanol ester-containing foods in both normocholesterolemic and hypercholesterolemic adults, as well as in children.
As with adverse interactions between pharmaceutical active ingredients, the same should be avoided between different active ingredients or nutrients, either pharmaceutical or dietary, that may be utilized or that are required to address the health needs of CVD and other health conditions as described above. Such interactions may reduce the effectiveness or intake of one or more of the ingredients used in the treatment. On some cases, the interaction of the ingredients used in the treatment of said conditions may cause the reduction of the effectiveness or intake of other dietary nutrients or ingredients, needed by the individual to maintain his general health and to be able to cope with the specific risk factors for which he is being treated for. In general, the observed adverse effect of phytosterol administration on the intake of vitamin A and beta-carotene, as well as of vitamin E and D, may also be relevant to other dietary nutrients, active pharmaceutical ingredients or dietary active ingredients which are lipophilic as the carotenoids and tocopherols. Such lipophilic dietary nutrients, active pharmaceutical ingredients or dietary active ingredients that their intake or effectiveness may be further compromised by parallel administration of phytosterols include pharmaceutical anti-dyslipidemia ingredients, natural anti-oxidants, nutritional or active lipids, etc. Schemes 1 and 2 exemplify the chemical structure of several lipophilic active ingredients and dietary nutrients as well as of vitamin A and E.


Pharmaceutically active ingredients have been developed with the goal of controlling high levels of blood cholesterol, and are widely used in the treatment of populations at risk regarding the above described conditions. Perhaps the most popular approach relies on active ingredients of the statins chemical family, known to inhibit the biosynthetic pathway of cholesterol production, hence leading to lower levels of blood cholesterol levels. Other active ingredients used are bile acid-binding resins, cholesterol absorption inhibitors, combination cholesterol absorption inhibitors and statins, fibrates, and niacin.
A variety of synthetic anti-oxidants have been used for industrial and food purposes. Current methodology prefers the utilization of natural anti-oxidants, mainly derived from phyto-sources. Such plant derived anti-oxidants include lycopene, mainly found in red plants, especially tomatoes, and lutein. Other phyto-originated anti-oxidants include zeaxanthin and beta-carotene. All these anti-oxidants can be classified as highly lipophilic, which is a direct result of their common carotenoidic skeleton. Other anti-oxidants include those of the polyphenolic chemical characteristics. Such carotenoid, polyphenolic and other lipophilic natural anti-oxidants are all useful in the treatment of the above described conditions and their parallel administration to phytosterols should be beneficial.
Omega-3 long chain poly-unsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have been recognized as promoting cardiovascular health, partly due to the anti-hypertriglyceridemia activity. The US Food and Drug Administration (FDA) has announced on September 2004 that these fatty acids have earned a qualified health claim for the reduction of the risk of CHD by consumption of EPA- and DHA-containing foods or dietary supplements. Indeed, the consumption of these fatty acids can be beneficial to the populations suffering from the disorders or conditions discussed above or to populations wishing to prevent or inhibit the onset of said conditions.
All these ingredients, as well as others, may be used, in combination or alone, in order to address the risk factors of CVD, CHD, metabolic disordered patients, high risk populations, cancer patients, etc.
Other lipophilic pharmaceutical active ingredients, as well as other lipophilic dietary supplements, mainly phyto-ingredients extracted or derived from different plant sources, are to be found or are under development to address different health conditions which require or may benefit from parallel administration or intake of phytosterols and/or their derivatives, and hence face the risk of their intake being compromised by phytosterol administration, as mentioned above.
Therefore, in light of the beneficial effects of phytosterols as a method of treatment of, for example, hypercholesterolemia, or, in combination with additional ingredients addressing a variety of CVD, CHD, metabolic disorders-associated health risks, including hypertriglyceridemia, hyperglycemia and oxidative stress, as well as other health conditions, such as, inter alia, cancer and immune deficiency-associated diseases as detailed below, it is a purpose of the invention to provide a method of treatment that will enable making the most of the beneficial effects of phytosterols/phytostanols and their esters, without the possible adverse effects of inhibition of intake of other important ingredients such as vitamins, anti-oxidants and also lipophilic pharmaceutical active ingredients.
Overweight or obesity substantially increases the risk of morbidity from a number of conditions, including type 2 diabetes mellitus (DM), hypertension, dyslipidemia, coronary heart disease, congestive heart failure, stroke, gallbladder disease, hepatic steatosis, osteoarthritis, sleep apnea, and endometrial, breast, prostate, and colon cancers. An increase in all-cause mortality is also associated with higher body weights. Many of these conditions can be treated or prevented by the use of phytosterols/phytostanols and their esters.
It is a further purpose of the invention to provide a method of treatment providing the beneficial effects of phytosterols/phytostanols and their esters on hypercholesterolemia while also controlling, and even promoting a reduction in a patient's body weight.
Thus, it is an object of the present invention to provide a method of treatment for conditions which require phytosterol therapy without adversely affecting the bioavailability of any one of lipophilic vitamins, lipophilic drugs, lipophilic nutrients, or any lipophilic pharmaceutically active ingredient, through the administration of a mixture of PS-E (phytosterol ester(s)) and DAG (mainly 1,3 diglyceride(s)), dissolved or dispersed in an edible oil or fat, or a composition or food article comprising the same, to a subject in need.
It is a particular object of the present invention to provide a method for increasing and/or maintaining the bioavailability and patient's serum levels of lipophilic vitamins, lipophilic drugs, lipophilic nutrients, or lipophilic pharmaceutically active ingredients, in a subject in need of phytosterol therapy through the administration of a mixture of PS-E (phytosterol ester(s)) and DAG (mainly 1,3 diglyceride(s)), dissolved or dispersed in an edible oil or fat, or a composition or food article comprising the same.
It is a further object of the present invention to provide a method for delivering phytosterol to a subject in need without adversely affecting the bioavailability of lipophilic substances such as lipophilic vitamins, lipophilic drugs, lipophilic nutrients, or any lipophilic pharmaceutically active ingredient increasing and/or maintaining the bioavailability and patient's serum levels of lipophilic vitamins, lipophilic drugs, lipophilic nutrients, or lipophilic pharmaceutically active ingredients, and with even increasing the serum level of said lipophilic substance, through the administration of a mixture of PS-E (phytosterol ester(s)) and DAG (mainly 1,3 diglyceride(s)), dissolved or dispersed in an edible oil or fat, or a composition or food article comprising the same.
It is a further object of this invention to provide a platform for the delivery of phytosterol and/or derivatives thereof to a subject without adversely affecting the bioavailability of lipophilic vitamins or other essential nutrients and drugs, as was known for conventional phytosterol preparations.
These and other objects of the invention will become apparent as the description proceeds.